Tissue engineering has shown promise toward creating blood vessel substitutes for cardiovascular surgery. Recent clinical studies suggest that tissue engineered vascular grafts fabricated from cell sheets (without exogenous scaffold materials), may offer advantages over scaffold-based grafts. However, the lengthy process required for their fabrication (~28 weeks), currently limits their use. In addition, the scaffold-free approach relies on the ability of cultured cells to produce an extracellular matrix (ECM) with sufficient strength and compliance for implantation. There have been few systematic studies in three dimensional tissue constructs of conditions that promote tissue growth, stimulate ECM synthesis, and improve mechanical strength due to the length of time required to generate tissue engineered vascular grafts. The overall goal of the proposed study is to 1) develop a rapid and inexpensive method to generate cell-derived, scaffold-free tissue constructs, and 2) evaluate the versatility of this method for assessing the effects of soluble factors and exogenous scaffolds on vascular graft structure, ECM synthesis, and mechanical function. The first Specific Aim is to develop a rapid and simple method to generate cell-derived tissue rings by seeding smooth muscle cells (SMC) into custom, non-adhesive, circular wells. Cell-derived tissue rings will then be subjected to histological analysis and mechanical testing to assess tissue structure and mechanical properties. In Specific Aim 2, cell-derived tissue rings will be generated using conditions established in SA1, and compared to rings fabricated from cells in collagen or fibrin gels, or cultured in growth-factor supplemented or serum-free media. The goal of SA2 is to assess the efficacy of the tissue ring system to measure the effects of soluble factors and exogenous scaffolds on engineered tissue composition and mechanics. If successful, the simplified system we propose would allow rapid fabrication of vascular tissue constructs for mechanical testing, accelerate cell-based vascular graft development, and facilitate discovery of factors that control tissue mechanical properties. PUBLIC HEALTH RELEVANCE: Tissue engineering has shown promise toward creating blood vessel substitutes to treat patients requiring vascular grafts for cardiovascular surgery and dialysis access. This proposal seeks to accelerate development and improve the fabrication process of biological vascular grafts for human transplantation.